Adenosine 5'-triphosphate (ATP) has many different physiological functions in the cell. For example, ATP is the energy source for many biochemical reactions, a precursor for ribonucleic acid (RNA) synthesis, the precursor for cyclic AMP synthesis, etc. ATP also functions as an extracellular messenger in neuronal and non-neuronal tissues. Extracellular ATP exerts its effects on these tissues by acting through membrane-associated purinoreceptors (Burnstock, G. Ann. NY Acad. Sci. (1990) 603:1-17). The purinoreceptors can be either ligand-gated ion channels (Bean, B. P. (1992) Trends Pharmac. Sci. 12:87-90; Bean, B. P. and Fried, D. D. (1990) Ion Channels 2:169-203) that are generally referred to as P.sub.2X receptors, (but also known as: purinergic channels, P.sub.2X R-channels, and ATP-gated channels) or G-protein-coupled (P.sub.2Y or P.sub.2V) receptors (Barnard, E. A. et al. (1994) Trends Pharmac. Sci. 15:67-70).
ATP-gated P.sub.2X receptors have been identified on autonomic and sensory neurons and on smooth muscle cells where they mediate membrane depolarization and, in certain cases, Ca.sup.2+ flux (Bean, B. P., supra). The P.sub.2X receptors are present on both nerve terminals and cell bodies of peripheral (PNS) and central (CNS) neurons. The receptors have short latency and inactivation time-constants that are consistent with a physiological role in neurotransmission. Their function includes fast signaling across synapses of the PNS and CNS. In the CNS, where ATP acts as a fast excitatory synaptic transmitter at nerve--nerve synapses, P.sub.2X receptors may mediate rapid sensory, motor, and cognitive functions. ATP may also serve as a co-transmitter of acetylcholine, substance P, and noradrenaline signals.
Peripheral P.sub.2X receptors also regulate effector structures such as cardiac and smooth muscle (Burnstock, G., supra), and are responsible for sympathetic vasoconstriction in small arteries and arterioles (Burnstock, G. (1988) Trends Pharmac. Sci. 9:116-7; Evans, J. R. and Surprenant, A. (1992) Br. J. Pharmacol. 106:242-9). In muscle and other cells, .sub.P2X receptors exert effects via direct conductance of Ca.sup.2+ ions as well as via conductance of Na.sup.+ ions; Na.sup.+ conductance causes membrane depolarization and subsequent activation of voltage-gated neuronal Na.sup.+ channels. Ca.sup.2+ influx through ATP-gated channels may also have a role in neurosecretory processes where the effector is a gland.
P.sub.2X receptors also have several roles in immune system responses. A purinoreceptor, originally designated P.sub.2Z, has been cloned and identified as a member of the P.sub.2X family (Surprenant, A. et al. (1996) Science 272:735-738). This P.sub.2X receptor, expressed in both rat brain and macrophages, is responsible for ATP-dependent lysis of antigen-presenting macrophages. ATP-gated channels are involved in mitogenic stimulation of human T-cells, and extracellular ATP exerts a synergistic effect on DNA synthesis stimulated by T-cell-specific mitogens such as phytohemagglutinin (Baricordi, O. R. et al. (1996) Blood 87:682-90). ATP can also mediate differentiation and cell death (apoptosis) of thymocytes and peripheral T cells (Chused, T. M. et al. (1996 J. Immunol) 157:1371-80). Other effects of extracellular ATP include mitogenesis of vascular smooth muscle cells (Erlinge, D. et al. (1995) Eur. J. Pharmacol. 289:135-49), and inhibition of platelet aggregation and clot size (Soslau, G. et al. (1995 Biochim. Biophys. Acta) 1268:73-80).
Ion channels gated by extracellular ligands such as nicotinic acid, serotonin, GABA, glycine or the excitatory amino acids all share common features and topology. Because of the functional similarities between those receptors and the ATP-gated P.sub.2X receptors, there was an expectation that the latter would also share common structural features. Cloning of P.sub.2X receptors from rat (Valera, S. et al.(1995) Nature 371:516-9; Brake, A. J. et al, supra) and human (Valera, S. et al.(1995) Recept. Channels 3:283-9; Longhurst, P. A. et al.(1996) Biochim. Biophys. Acta 1308:185-8) demonstrates that the P.sub.2X receptors comprise a separate and distinct family of ion channels. However, the sequences of cloned P.sub.2X cDNAs (Valera, S. et al, Nature, supra; Brake, A. J. et al, supra) have homology to the RP-2 partial cDNA clone previously identified by Owens G. P. (1991; J. Molec. Cell Biol. 11:4177-88). The RP-2 gene is activated in thymocytes which have been induced to undergo apoptosis. Such activation of RP-2 is consistent with a role for ATP and P.sub.2X receptors in programmed cell death.
Numerous P.sub.2X receptors have been cloned from the rat and are distinguishable pharmacologically by their responses to various ATP analogs such as .alpha.,.beta.-methylene-ATP, .beta.,.gamma.-methylene-ATP, 2-methyl-thio-ATP, and .gamma.-thio-ATP. For example, when cloned rat P.sub.2X receptor g558831 (SEQ ID NO:5) is expressed in Xenopus oocytes, 2-methyl-thio-ATP and .gamma.-thio-ATP are roughly equivalent agonists, whereas .alpha.,.beta.-methylene-ATP and .beta.,.gamma.-methylene-ATP are inactive as either agonists or antagonists ( Brake, A. J. et al.(1995) Nature 371:519-23). This profile is similar to that of native P.sub.2X receptors expressed by rat PC12 cells and some sensory and autonomic neurons (Nakazawa, K. et al.(1990) J. Physiol. 428:257-72; Majid, M. A. et al.(1992) Biochim. Biophys. Acta 1136:283-289; Fieber, L. A. and Adams, D. J. (1991) J. Physiol. 434:239-56; Cloues, R. et al.(1993) Pflug. Archiv 424:152-8), but differs from that of P.sub.2X receptors expressed by vascular smooth muscle, vas deferens, and some CNS neurons (Friel, D. D. (1988) J. Physiol. 401:361-80; Evans, R. J. et al.(1992) Nature 357:503-5; Benham, C. D. and Tsien, R. W. (1987) Nature 328:275-8).
At the molecular level, the P.sub.2X proteins are of variable length and have no significant homology with receptors outside of the family. The lack of an apparent amino terminal signal sequence suggests that both the N- and C-termini of the protein are cytoplasmic. There is a conserved loop, bounded by two hydrophobic putative transmembrane domains, that contains regularly spaced hydrophilic cysteine residues and likely resides on the outside of the cell membrane; all three potential N-linked glycosylation sites are within this loop. Extracellular ATP may bind to the loop in region(s) that resemble Walker type-A phosphate binding motifs (Saraste, M. et al.(1990) Trends Biochem. Sci. 15:430-4).
Northern blot analyses detect P.sub.2X messenger RNAs of varying sizes and relative abundances in adrenal gland, bladder, brain, intestines, lung, ovary, pituitary, retina, spinal cord, spleen, testis, thymus, and vas deferens, but fail to detect related sequences in liver or kidney (Valera, S. et al. Nature, supra; Brake, A. J. et al, supra.
The discovery of polynucleotides encoding novel human P.sub.2X purinoreceptors, and the molecules themselves, present the opportunity to investigate ATP-gated signal transduction. Discovery of molecules related to P.sub.2X purinoreceptors satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the diagnosis, prevention, and treatment of disorders and diseases of the immune system, nervous system, cardiovascular system, and of smooth muscle.